| Objective:Osteoarthritis is a chronic degenerative disease of joints,mainly due to aging,trauma,obesity,joint deformity or congenital joint abnormalities and other factors.The pathogenic factors are often not single,but often a combination of many factors.The pathological changes of osteoarthritis mainly include progressive destruction of articular cartilage,synovial hypertrophy,chronic inflammatory hyperplasia of synovial cells,reactive hyperplasia of subarticular cartilage and so on.This disease is mainly concentrated in the middle-aged and elderly people over 50,especially in the knee joint,hip joint and other joints with large weight bearing degree,and the knee joint is the most common clinically.These joints are subjected to excessive stress over a long period of time and can eventually lead to degenerative damage.The main clinical symptoms are chronic joint pain,joint swelling,joint stiffness,limited mobility and even joint deformity resulting in disability and so on.The reasons may be closely related to estrogen,aging,weight gain,excessive joint burden and other factors.According to the statistics of WHO in 1990,knee osteoarthritis ranks 11th in the disability spectrum,and it keeps rising,and is now the second highest disability rate in the world.Statistics have shown that knee osteoarthritis affects as many as 400 million people worldwide.In China,the prevalence of knee osteoarthritis is as high as half of the elderly population over 60 years old,and 80%of the elderly population over 75 years old.The incidence of osteoarthritis is not only high but also high in the elderly.Knee osteoarthritis in China’s middle-aged and elderly people over 50 years old,the disability rate is extremely high,has been more than half.The harm degree of chronic pain and limb dysfunction caused by osteoarthritis has been greater than that of cardiovascular and cerebrovascular diseases.Due to the lack of early diagnosis and monitoring the disease development of specific biomarkers and drug targets,has been in the middle-late stage when diagnosed,most of the patients medical treatment are not beautiful,seriously affecting the quality of life,in the end line of joint replacement surgery is needed to eradicate all symptoms,patients families and society to bring tremendous psychological pressure and economic burden.Therefore,for patients with osteoarthritis,it is of great significance to improve the success rate of early diagnosis and find molecular therapeutic targets at the same time.In this paper,bioinformatics methods were used to screen the differential genes of osteoarthritis,and CXCL2 was selected as the target gene to study its expression in osteoarthritis patients and normal people,as well as its effect on the proliferation and apoptosis of osteoarthritis synovial cells and the possible pathway mechanism.Methods:1.Bioinformatics method was used to screen differential genes of osteoarthritis,and CXCL2 was selected as the target gene.2.Western blot,qRT-PCR was used to detect CXCL2 expression in synovial tissues of human osteoarthritis and normal bone and joint synovial tissues.3.Human normal fibroblast synovial cell lines（HFLS）were induced with four concentrations of il-1β（0,2,5,10 ng/ml）for 24h to establish an inflammatory model.Finally,il-1β（10ng/ml）was selected to establish an inflammatory cell line.Western blot was used to detect the expression of il-6,an inflammatory indicator.CCK8 was used to detect cell viability.4.Western blot was used to detect the expressions of CXCL2,COX-2,MMP-13,P38,P-JNK,P-IKBa in IL-1β（10ng/ml）induced synovitis model.5.Transfected cell lines were established,Western blot and qRT-PCR were used to detect CXCL2 infection,and fluorescence microscope was used to detect the infection efficiency.6.Four groups were set up in the follow-up experiment,（1）normal cell group（NC group）;（2）IL-1β+siRNA-CXCl2 group;（3）IL-1β+Si-NC group;（4）IL-1βgroup.Proliferation of synovial cells was detected by CCK8 and apoptosis was detected by flow cytometry with Annexin V/PI staining.7.Western blot was used to detect the expression of MAPK pathway and NF-KB pathway related proteins in four groups.Results:1.GSE55235 gene chip data analyzed 10 cases of bone arthritis synovial tissue samples and 10 cases of normal synovial tissue samples,to|log2FC|>1 is the screening criteria,select 587 osteoarthritis differentially expressed genes,gene expression differences of421 high,low gene expression differences of 166.Heat maps showed that CXCL2 was significantly overexpressed in the osteoarthritis samples compared with healthy controls（P<0.05）,and KEGG pathway analysis showed that its mechanism acted on MAPK and NF-KB pathways.2.The expression of CXCL2 m RNA in synovial tissue of human osteoarthritis was2.1±0.31 times that in normal human osteoarthritis（P<0.01）.The expression level of CXCL2 protein in synovial tissues of human osteoarthritis was 2±0.28 times higher than that in normal synovial tissues（P<0.05）,suggesting that the expression levels of CXCL2m RNA and protein in synovial tissues of human osteoarthritis were significantly higher than that in normal synovial tissues.3.IL-1β（0,2,5,10 ng/ml）was used to induce inflammation model,and the expression of IL-6 was detected by western blot.The expression of IL-6 increased progressively at0ng/ml,2ng/ml,5ng/ml,and 10ng/ml（P<0.001）.CCK8 showed that the cell activity was the lowest at 10ng/m L IL-1β（P<0.001）.4.Western blot showed that the expressions of CXCL2,COX-2,P-P38,P-JNK and P-IKBa in IL-1β-induced synovial inflammation model were significantly higher than those in normal synovial cells（P<0.05）.5.The infection efficiency of IL-1β+siRNA-CXCl2 group was 90%±2.1%,and that of IL-1β+Si-NC group was 83%±2.4%.The protein expression level of CXCL2 in IL-1β+siRNA-CXCl2 group was significantly lower than that in IL-1βgroup and IL-1β+Si-NC（P<0.01）.The m RNA expression level of CXCL2 in IL-1β+siRNA-CXCl2 group was significantly lower than that in IL-1βgroup and IL-1β+Si-NC group（P<0.05）.6.CCK8 showed that the proliferation rate of IL-1β+siRNA-CXCl2 group was significantly lower than that of IL-1βgroup and IL-1β+Si-NC group（P<0.01）.The apoptosis rate of THE IL-1β+siRNA-CXCl2 group was 6.4%,that of the IL-1βgroup was 3.9%,that of the IL-1β+Si-NC group was 4.6%,and that of the NC group was9.1%.The apoptosis rate of IL-1β+siRNA-CXCL2 group was significantly higher than that of IL-1βgroup and IL-1β+Si-NC group（P<0.01）.7.The protein expression levels of phosphorylated P38,P65 and IKBa in THE IL-1β+siRNA-CXCL2 group were significantly lower than those in the IL-1βgroup and the IL-1β+Si-NC group（P<0.05）,while the protein expression levels of total P38 and P65were not significantly different among the four groups.Conclusion:1.The expression of CXCL2 in synovial tissues of human osteoarthritis was significantly higher than that of normal synovial tissues.CXCL2 is a pro-inflammatory gene.2.Il-1βinduces osteoarthritis up-regulation of CXCL2,osteoarthritis effector factors COX-2,MMP-13 and inflammatory index IL-6 in human fibroblastsynovial cells through the activation of MAPK and NF-KB signaling pathways.3.Inhibition of CXCL2 expression can enhance the ability of osteoarthritis synovial cells to resist proliferation and promote apoptosis by inhibiting MAPK and NF-KB signaling pathways. |
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